Project Summary Filoviruses cause severe hemorrhagic fevers and are among the deadliest human viruses with no approved treatment or prophylactic options. Periodic outbreaks, such as the recent epidemic of Ebola virus (EBOV) in West Africa, exhibit mortality rates ranging from 50-90%. They are classified as ?Category A priority bioweapon agents? by the Centers for Disease Control and Prevention. The most advanced candidates for anti-filovirus therapy with proven efficacy in non-human primates (NHPs) utilize RNA interference (RNAi), high dose mixtures of monoclonal antibodies (mAbs), or repurposed influenza, herpes or hepatitis C inhibitors. Although they represent important advances, these approaches have serious limitations including the high therapeutic doses required (20-30 g of mAb/patient), limited spectrum (primarily Zaire strain) and/or narrow therapeutic windows (small molecules/RNAi). Building on recent work demonstrating the critical nature of endosomal interactions between the filoviral glycoprotein (GP) and the host cholesterol transport protein Niemann-Pick C1 (NPC1) for filoviral entry, we have developed small molecules and antibodies that disrupt this mechanism to inhibit viral entry. These include several bispecific GP-targetomg pan-ebolavirus broadly neutralizing antibodies (bNAbs) efficacious in multiple animal models, with down-selection to a final cocktail based on NHP efficacy underway. In parallel, we identified an NPC1-binding phenylpiperazine (MBX-3587), which inhibits the GP-NPC1 interaction to block infection by virulent EBOV and MARV at low to sub M concentrations in vitro, demonstrates excellent PK/PD properties, exhibits excellent murine tolerability, has established efficacy in mice, and is currently being optimized for a targeted IND. In this proposal we will tether MBX-3587 to bispecific pan-ebola bNABs to target broad-spectrum antibody-drug conjugates (ADCs) that synergistically inhibit viral entry. The objective of this proposal is to demonstrate that these ADCs will provide improved efficacy and selectivity for filovirus treatment over either agent alone, providing highly efficacious pan-ebola agents, broadening the therapeutic window and easing drug stockpiling efforts. We will accomplish this objective through three specific aims: In Aim 1 we will synthesize and complete physical characterization of >40 ADCs demonstrating serum stability with efficient endosomal release. In Aim 2 we will prioritize ADCs through in vitro analysis of pan-ebola inhibitory activity. In Aim 3 we prioritize ADCs for PK and toxicity and evaluate two pan- ebola ADCs for in vivo efficacy in a mouse model of EBOV infection.